Evaluation of the LightCycler Methicillin-Resistant Staphylococcusaureus (MRSA) Advanced Test for Detection of MRSA NasalColonization

W. C. Yam,a Gilman K. H. Siu,a P. L. Ho,a T. K. Ng,b T. L. Que,c K. T. Yip,c Cathie P. K. Fok,c Jonathan H. K. Chen,a Vincent C. C. Cheng,a

K. Y. Yuena

Department of Microbiology, Queen Mary Hospital, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, Chinaa;Pathology Department, Princess Margaret Hospital, Hong Kong Special Administrative Region, Chinab; Department of Clinical Pathology, Tuen Mun Hospital, Hong KongSpecial Administrative Region, Chinac

Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization is crucial for the prevention and con-trol of MRSA infections in health care settings. The LightCycler MRSA Advanced Test (Roche Diagnostics) is a commerciallyavailable real-time PCR assay for direct detection of MRSA nasal colonization by targeting of the staphylococcal cassette chro-mosome mec (SCCmec)-orfX junction. The diagnostic performance of the assay was compared with that of ChromID MRSA agar(bioMérieux) culture and an in-house duplex real-time PCR assay. Among 1,246 nasal swab specimens collected from 2 generalhospitals in Hong Kong, 174 (14%) were considered true positive for MRSA. Chromogenic culture and the in-house real-timePCR assay identified 147 (84.5%) and 133 (76.4%) true-positive cases with specificities of 100% and 98.6%, respectively. Based onthe target melting temperature (Tm) values (57.0 to 62.0°C) defined by the manufacturer, the LightCycler MRSA Advanced Testidentified only 85 (48.9%) true-positive specimens. Interestingly, an additional 60 (34.5%) true-positive specimens were detecteddespite atypical Tm values of 55°C, providing overall sensitivity and specificity values of 83.3% and 99%, respectively. Amongisolates with Tm values of 55°C, most were typed as clonal complex 45 (CC45). By sequence analysis of the SCCmec-orfX junction,characteristic single-nucleotide polymorphisms (SNPs) were identified only in isolates with Tm values of 55°C and not in thosewith typical Tm values. It is conceivable that those SNPs were located inside the target region of the proprietary hybridizationprobes, which resulted in a Tm shift in the melting curve analysis. Our study highlights the importance of a global evaluation ofcommercial kits so that the interpretation algorithm covers different lineages of MRSA clones prevalent in various geographicalregions.

Methicillin-resistant Staphylococcus aureus (MRSA) has beenestablished as a health care-associated pathogen in many

countries worldwide since the 1980s (1, 2). Reports of MRSA car-riage or acquisition in the community have become a major focusof medical and public concern during the past decade (3, 4). Nasalcolonization is known to be a risk factor for the development ofMRSA-associated disease (5). Rapid diagnosis of MRSA nasal col-onization facilitates early implementation of control measures toprevent ongoing infection and transmission (6). The LightCyclerMRSA Advanced Test (Roche Diagnostics, Basel, Switzerland) isone of the commercially available real-time PCR assays that aredesigned for direct detection of MRSA nasal colonization by tar-geting of the genetic region between staphylococcal cassette chro-mosome mec (SCCmec) and the S. aureus chromosomal orfX gene,based on hybridization probe technology. According to the prod-uct information, the range of melting temperature (Tm) valuesobserved in evaluation studies with various epidemic MRSAclones was 57.0 to 62.0°C. However, because of the enormoussequence diversity of SCCmec, MRSA clones with rarely encoun-tered SCCmec types might yield abnormal Tm values or even false-negative signals. A previous study reported a single-nucleotidepolymorphism (SNP) in the SCCmec-orfX junction found inMRSA clonal complex 398 (CC398), resulting in a shift of the Tm

value to 55°C in the LightCycler MRSA Advanced Test (7). Al-though the result could still be manually interpreted as “positivefor MRSA DNA (presumed MRSA nasal colonization)” accordingto the manufacturer’s guidelines, the calculation algorithms em-

bedded in the automated assay interpretation software (MicroAnalysis Software) of the LightCycler MRSA Advanced Test re-ported the result as “MRSA result: not detected,” with a specificcomment of “peak(s) outside target TM range” (7).

The LightCycler MRSA Advanced Test has been introduced in2 general hospitals in Hong Kong since 2010 for the intendedpurpose of direct detection of MRSA in nasal swabs. This studyaimed to compare the diagnostic performance of the LightCyclerMRSA Advanced Test with that of ChromID MRSA agar cultureand an in-house MRSA duplex real-time PCR assay for the detec-tion of MRSA clones that are commonly circulating in HongKong.

MATERIALS AND METHODS

Specimen collection. Between December 2010 and December 2011, atotal of 1,246 nonduplicated nasal swabs were collected from 2 generalhospitals in Hong Kong. The subjects included patients in hospital inten-

Received 19 February 2013 Returned for modification 19 March 2013Accepted 16 June 2013

Published ahead of print 19 June 2013

Address correspondence to W. C. Yam, wcyam@hkucc.hku.hk.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.00488-13

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sive care units (ICUs), non-ICU settings, or dialysis units and patientswho were admitted from nursing homes.

Molecular methods. The swab heads were added to sample prepara-tion buffer for swab extraction and mechanical lysis using a MagNA Lyserinstrument (Roche Diagnostics), according to the manufacturer’s guide-lines. The LightCycler MRSA Advanced Test also was performed accord-ing to the manufacturer’s instructions.

The in-house MRSA duplex real-time PCR assay was designed to tar-get a segment of the mecA gene and the S. aureus-specific genomic frag-ment sau. A total of 5 �l of the remaining DNA extract was added to thePCR mixture containing 1� LightCycler TaqMan Master reaction mix-ture (Roche Diagnostics, Germany), 0.5 �M primers for sau and mecA(sau-F, 5=-GACCTAATTGCTGCAACTGATCG-3=; sau-R, 5=-ACTCGTGGTGGTCATATGGAAGC-3=; mecA-F, 5=-GCCAATTCCACATTGTTTCG-3=; mecA-R, 5=-ACGGTAACATTGATCGCAACG-3=), and 0.2 �MTaqMan probes for sau and mecA (sau-P, 5=-6-carboxyfluorescein-AAATCTTCAGCATTATGAATAAATTCGTAACG-black hole quencher 1[BHQ1]-3=; mecA-P, 5=-Yakima yellow [YAK]-TTACCACGTTCTGATTTTAAATTTTCAATATGTATGC-BHQ1-3=). Real-time PCRs were car-ried out in a LightCycler 2.0 system (Roche Diagnostics, Germany) underthe following conditions: initial denaturation at 95°C for 10 min and 40cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s. The thresholdcrossing point (Cp) value for both sau and mecA targets was defined as 38cycles by a receiver operating characteristic (ROC) curve with maximalarea under the curve (AUC) and Jouden index. Samples with cycle num-bers of �38 were considered to have positive amplification. Samples withCp values of �38 cycles were required to be retested by real-time PCRassay. If the retest showed a Cp value of �38 cycles, then the sample wasconsidered to have positive amplification. Alternatively, samples with cy-cle numbers of �38 in retests were regarded as negative. To minimize therisk of false-positive results caused by nasal cocolonization with methicil-lin-resistant coagulase-negative staphylococci (MRCNS) and methicillin-susceptible Staphylococcus aureus (MSSA), real-time PCR assays wereconsidered positive for MRSA only when both sau and mecA targetsshowed positive amplification, with a difference in Cp values (�Cp) of �3cycles.

Culture methods. The swab heads were streaked onto ChromIDMRSA agar (bioMérieux, France) directly before they were cut and addedto the sample preparation buffers, which were used for DNA extractionfor both LightCycler MRSA Advanced Test and in-house duplex real-timePCR analyses. The plates were incubated for up to 48 � 4 h at 37°C. Mauvecolonies obtained after 24 to 48 h of incubation were presumed to beMRSA, followed by confirmation using Gram staining, latex agglutination(Slidex Staph Plus; bioMérieux, France), and tube coagulase testing.

Definition of true-positive MRSA results. Samples were consideredtrue positive for MRSA when they showed positive results for culture onChromID MRSA agar. Culture-negative samples that showed positive re-sults in both molecular assays (i.e., LightCycler MRSA Advanced Test andin-house duplex real-time PCR assay) also were considered true positivefor MRSA.

Molecular typing of MRSA. Representatives of MRSA strains withdifferent Tm values in the LightCycler MRSA Advanced Test were ran-domly selected for further characterization by molecular typing methods,including S. aureus protein A gene (spa) typing and multilocus sequencetyping (MLST), using standard protocols (8, 9). Sequencing analysis of theSCCmec-orfX junction was performed to screen for SNPs, according tothe method described by Hagen et al. (10).

Statistical analysis. The results of direct ChromID MRSA agar cul-ture, the LightCycler MRSA Advanced Test, and the in-house duplexreal-time PCR assay were compared with each other. Statistical signif-icance was determined by the chi-square test. P values of �0.05 wereconsidered statistically significant. The diagnostic performance pa-rameters (sensitivity, specificity, positive predictive value [PPV], andnegative predictive value) of the three assays also were determined,and the results were expressed as percentages, with 95% confidence

intervals, using an adjusted Wald method. For the in-house real-timePCR assay, Cp values for false-positive samples were compared withthose for true-positive samples using the Mann-Whitney U test. Dif-ferences were considered statistically significant when P values were�0.05.

RESULTS

Among 1,246 nasal swabs, 174 (14%) were considered true posi-tive for MRSA based on the overall results from cultures and real-time PCR assays (Table 1). Direct culture on ChromID MRSAagar, which was considered the conventional method for MRSAdetection with 100% specificity, identified 147 (84.5%) true-pos-itive cases in this study (Table 2).

The in-house duplex real-time PCR assay showed positive am-plification for both sau and mecA targets in 166 specimens. How-ever, 18 of those were immediately considered negative in thein-house duplex real-time PCR assay as their �Cp values were �3cycles (range, 3.8 to 14.5 cycles). ChromID MRSA agar cultureand the LightCycler MRSA Advanced Test confirmed that none ofthe 18 samples was positive for MRSA, although cultures showedthe coexistence of MSSA and MRCNS in 7 of those samples.Among the 148 specimens that were positive in the in-house du-plex real-time PCR assay, 133 were true positive for MRSA and 15were considered false-positive cases, as they were negative in bothChromID MRSA agar culture and the LightCycler MRSA Ad-vanced Test. The cultures of 6/15 false-positive samples showedpositive results for MRCNS, with one showing coexistence withMSSA. All remaining false-positive samples had negative cultureresults. False-positive samples were found to have greater Cp val-ues than true-positive samples (P � 0.008, Mann-Whitney Utest). The diagnostic sensitivity and specificity of the in-house du-plex real-time PCR assay were 76.4% (133/174 samples) and98.6% (1,057/1,072 samples), respectively (Table 2).

A total of 92 specimens were positive in the LightCyclerMRSA Advanced Test, with Tm values of 59°C (within the rangeof 57 to 62.0°C suggested by the embedded Micro AnalysisSoftware). Only 85 of those specimens were true-positiveMRSA cases, which resulted in a low diagnostic sensitivity of48.9% (85/174 samples). Interestingly, another 64 samplesshowed positive amplification of the SCCmec-orfX junction,with atypical Tm values of 55°C in the melting curve analysis.The same results were obtained when the test was repeated withthe corresponding cultured isolates. Among those samples, 60were considered true positive for MRSA, as indicated either byculture followed by biochemical tests or by positive resultsfrom our in-house duplex real-time PCR assay. Regardless ofthe postamplification Tm values, overall the LightCycler MRSAAdvanced Test identified 145 true-positive MRSA specimensby amplifying the SCCmec-orfX junction. Among 11 false-pos-itive cases, 3 demonstrated MRCNS and 8 showed negativeresults. The overall sensitivity and specificity of the LightCyclerMRSA Advanced Test were 83.3% (145/174 samples) and99.0% (1,061/1,072 samples), respectively (Table 2).

A total of 35 and 24 specimens that showed Tm values of 55°Cand 59°C, respectively, in the LightCycler MRSA Advanced Testwere randomly selected for further characterization (Table 3). Ofthe isolates with Tm values of 55°C, a majority (32/35 isolates[91%]) were typed as clonal complex 45 (CC45) sequence type 45(ST45), with spa patterns of t1081 (n � 23), t1857 (n � 2), t7317(n � 2), t7854 (n � 2), t1768 (n � 1), and t8404 (n � 1). One

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CC45 isolate had an unknown spa type due to repeated PCR fail-ure (Table 3). Among the 32 CC45 strains, 23 (72%) were isolatedfrom patients admitted from nursing homes, whereas the restwere evenly distributed among ICU (n � 3), non-ICU (n � 3),and dialysis unit (n � 3) patients. The remaining 3 strains with Tm

values of 55°C belonged to CC398 (ST398, n � 2) or CC672(ST672, n � 1). All of those strains were isolated from patients innon-ICU wards. The strains characterized by Tm values of 59°Cwere typed to 4 clonal complexes, i.e., CC5 (ST5, n � 3), CC8(ST8, n � 2; ST239, n � 2), CC9 (ST9, n � 1), and CC22 (ST22,n � 16) (Table 3). CC22 strains were isolated mainly from ICU

(n � 6) and non-ICU (n � 6) wards, whereas the other identifiedlineages were evenly distributed among the collection sites.

By systematic sequencing of the SCCmec-orfX junction, allCC45 MRSA strains were found to have identical sequences inmultiple alignments (Fig. 1). A detailed investigation of the se-quences revealed some polymorphisms that are uncommonamong S. aureus GenBank sequences; one characteristic SNP,C¡T at position 417, was found exclusively in all investigatedCC45 MRSA strains. Another SNP, T¡C at position 441, wasidentified in all selected MRSA strains with Tm values of 55°Cexcept for CC672, which harbored another unique SNP, G¡A at

TABLE 1 ChromID MRSA agar culture, LightCycler MRSA Advanced Test, and in-house duplex real-time PCR assay results

True MRSAresult (n)a

Direct ChromID MRSAagar culture resultb,c

Real-time PCR results for:

No. of samples(total n � 1,246)

LightCycler MRSA AdvancedTest atb,d:

In-house duplex(sau � mecA) assayc,d57–62°C 55°C

Positive (174) Positive Positive Negative Positive 56Positive Positive Negative Negative 13Positive Negative Positive Positive 42Positive Negative Positive Negative 7Positive Negative Negative Positive 5Positive Negative Negative Negative 20Positive Invalide Invalide Positive 3Positive Invalide Invalide Negative 1Negative Negative Positive Positive 11Negative Positive Negative Positive 16

Negative (1,072) Negative Negative Negative Positive 15Negative Negative Positive Negative 4Negative Positive Negative Negative 7Negative Negative Negative Negativef 1,046

a Samples with positive results for ChromID MRSA agar culture and samples with positive results in both the LightCycler MRSA Advanced Test and the in-house duplex real-timePCR assay were considered true positive for MRSA.b P � 0.001 for culture versus LightCycler MRSA Advanced Test results.c P � 0.001 for culture versus in-house duplex assay results.d P � 0.001 for LightCycler MRSA Advanced Test versus in-house duplex assay results.e The results were defined as invalid because of a failure of internal control amplification due to the presence of inhibitors.f A total of 18 samples with positive amplification for both sau and mecA genes were considered negative in the in-house duplex PCR assay because their �Cp values were �3 cycles.

TABLE 2 ChromID MRSA agar culture, LightCycler MRSA Advanced Test, and in-house duplex real-time PCR assay performance in detection ofMRSA

Assay

No. of samples with the indicated resulta Performance (% [95% CI])h

Positive NegativeTruepositiveb

Falsepositive

Falsenegative

Truenegativec Sensitivity Specificity

Positivepredictive value

Negativepredictive value

Direct ChromID MRSAagar culture

147 1,099 147 0 27 1,072 84.5 (78.3–89.2) 100 (99.7–100.0) 100 (97.8–100.0) 97.5 (96.4–98.3)

LightCycler MRSAAdvanced Test

156d 1,090 145e 11f 29 1,061 83.3 (77.0–88.5) 99.0 (98.2–99.5) 92.9 (87.7–96.1) 97.3 (96.2–98.2)

In-house duplex real-time PCR assay

148 1,098g 133 15 41 1,057 76.4 (69.6–82.2) 98.6 (97.7–99.2) 89.9 (83.9–93.9) 96.3 (95.0–97.3)

a A total of 1,246 samples were used to evaluate the performance of culture and real-time PCR assays for the detection of MRSA nasal colonization.b A total of 174 samples were considered true positive for MRSA based on the overall results of cultures and real-time PCR assays (also see Table 1).c A total of 1,072 samples were considered true negative for MRSA based on the overall results of cultures and real-time PCR assays (also see Table 1).d Among 156 positive samples from the LightCycler test, 92 showed Tm values of 59°C, whereas 64 showed Tm values of 55°C.e Among 145 true-positive samples indicated by the LightCycler test, 85 showed Tm values of 59°C, whereas 60 showed Tm values of 55°C.f The 11 false-positive samples from the LightCycler test included 7 samples with Tm values of 59°C and 4 samples with Tm values of 55°C.g A total of 18 samples with positive amplification for both sau and mecA genes were considered negative in the in-house duplex PCR assay because their �Cp values were �3 cycles.h Results were determined as percentages and 95% confidence intervals (CIs) by an adjusted Wald method.

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position 435. The previously described SNP (G¡A at position366) associated with livestock-associated MRSA CC398 (7) wasidentified in both of our CC398 MRSA strains. Among MRSAstrains with Tm values of 59°C, CC8 (ST8) harbored a SNP atposition 402, whereas CC22 showed a nucleic acid substitution atposition 354. All remaining selected isolates did not show anySNPs throughout the investigated SCCmec-orfX junction.

DISCUSSION

MRSA nasal colonization is a known risk factor for staphylococcalinfections, and carriers exhibit an increased risk of infection withthe strain they carry (11, 12). Direct culture from nasal swabs isconsidered the conventional standard method for detection ofMRSA nasal colonization. Despite very high specificity, the sensi-tivity of this method is always dependent on the quantities ofviable MRSA on the swab samples, which in turn depends onwhether the swab heads reached and sampled the particular nasalregions with high densities of viable MRSA. In our study, only84% of true-positive samples were detected by ChromID MRSAagar culture after 48 h of incubation. Similar results were reportedby Peterson et al. (13); in that study, the sensitivities of directlyplated cultures performed in 5 different evaluation centers rangedfrom 78.6% to 95.1%, indicating that considerable proportions offalse-negative results could be obtained from directly plated cul-tures without the supplement of molecular assays.

Recently developed PCR-based assays, either developed in-house or available commercially, provide rapid diagnosis (within2 h) of MRSA colonization in patients. Current PCR approachesare generally classified into two categories, (i) single detection ofthe SCCmec-orfX integration site or (ii) duplex PCR for coampli-fication of the mecA gene and a S. aureus-specific region such asnuc (12, 14), femA (15), or sau (16).

Most of the commercially available rapid tests, such as theLightCycler MRSA Advanced Test, the GeneXpert MRSA test(Cepheid, Sunnyvale, CA), and the GeneOhm MRSA assay (BD,Franklin Lakes, NJ), are based on detection of the SCCmec-orfXjunction by using one primer specific for different types ofSCCmec elements but not targeting the mecA gene specifically andanother primer specific for S. aureus chromosomal sequencesflanking the SCCmec integration site. Although these tests usuallyprovide high specificities (�95%), positive predictive values gen-

erally range from 72% to 78% (17–20). False-positive cases inEurope have been reported, as 4.6% of the investigated MSSAstrains were positive for SCCmec elements but lacked the mecAgene in a previous study (18). In the present study, 11 (7%) spec-imens that showed positive results with the LightCycler MRSAAdvanced Test were defined as false-positive cases. However,none of those was positive for MSSA as indicated by culture andlatex agglutination. A similar false-positive rate was reported byPeterson et al., who demonstrated that the LightCycler MRSAAdvanced Test and the BD GeneOhm MRSA assay showed false-positive rates of 6% and 25%, respectively (13).

It would be expected that MSSA with SCCmec elements couldbe resolved by amplification of the mecA gene together with a S.aureus-specific region. However, the specificity and positive pre-dictive value (PPV) of this method were usually compromised byMRCNS and MSSA nasal cocolonization. A previous study re-ported that MSSA and MRCNS cocolonization was detected for3.4% of nasal swab specimens and an unacceptable PPV of about40% was obtained with simultaneous detection of femA and mecA(21). The performance of this method was significantly improvedin a subsequent study, which showed that �Cp values for femAversus mecA were �3 cycles in more than 80% of true-positiveMRSA cases (15, 22). A recent study demonstrated that the Cpdifferences between SCCmec-orfX and mecA were �3 cycles formixtures of genomic DNA from MRCNS and MSSA (23). Theobservation was confirmed by our pilot study, which showed thatthe presence of MRSA could be distinguished from MSSA/MRCNS cocolonization on the basis of �Cp values of �3 cyclesfor sau versus mecA unless there were �10-fold differences inconcentrations between MSSA and MRCNS (data not shown). Inthis study, only one sample with MSSA/MRCNS cocolonizationshowed a �Cp value of �3, indicating that the differences betweenMSSA and MRCNS populations in the nasal cavity usually werelarge enough to be detected on the basis of �Cp values of �3,resulting in a PPV of about 92%. Nevertheless, given the highsequence similarities and the ability to exchange genetic materialamong staphylococcal species inside the nasal cavity, a false-pos-itive rate of 10% was noted for our in-house assay. False-positivesamples were found to exhibit greater Cp values than true-positivesamples (P � 0.008, Mann-Whitney U test), indicating that the

TABLE 3 Molecular characteristics of investigated MRSA strains with Tm values of 55°C and 59°C in the LightCycler MRSA Advanced Test

No. of testedstrains Tm (°C) spa type MLST type

Clonalcomplex SNPs in the SCCmec-orfX junction

23 55 t1081 ST45 CC45 C¡T at position 417, T¡C at position 4411 55 t1768 ST45 CC45 C¡T at position 417, T¡C at position 4412 55 t1857 ST45 CC45 C¡T at position 417, T¡C at position 4412 55 t7317 ST45 CC45 C¡T at position 417, T¡C at position 4412 55 t7854 ST45 CC45 C¡T at position 417, T¡C at position 4411 55 t8404 ST45 CC45 C¡T at position 417, T¡C at position 4411 55 PCR failure ST45 CC45 C¡T at position 417, T¡C at position 4412 55 t4677 ST398 CC398 A¡G at position 312, G¡A at position 318, G¡A at position 366,

T¡C at position 4411 55 t1309 ST672 CC672 G¡A at position 4353 59 t002 ST5 CC5 No SNP found2 59 t008 ST8 CC8 A¡T at position 4021 59 t899 ST9 CC9 No SNP found16 59 t032 ST22 CC22 G¡A at position 3542 59 t037 ST239 CC8 No SNP found

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“wrong results” were mainly attributable to samples with higherCp values, which might result from nonspecific amplification ofhomologous genetic regions in MRCNS.

Our study reported an unexpectedly large proportion ofMRSA-positive specimens with atypical Tm values of 55°C in theLightCycler MRSA Advanced Test. Most of these strains belongedto the CC45 lineage, which accounts for most MRSA colonizationin Hong Kong, especially among residents of nursing homes (20).In addition to nasal colonization, the CC45 lineage is significantlyassociated with the increasing multisusceptible MRSA isolatedfrom blood cultures in our region (24). In this study, we noted acharacteristic SNP in the SCCmec-orfX junction that was found inall investigated CC45 MRSA isolates. It was thought that this par-ticular mutated position was located inside the binding regions ofthe proprietary hybridization probes for the LightCycler MRSAAdvanced Test, resulting in a Tm shift from 59°C to 55°C. In ad-dition to CC45, MRSA CC398 and CC672, which harbor SNPs atdifferent nucleotide positions of the SCCmec-orfX junction, wereassociated with Tm values of 55°C. The three MRSA clones collec-tively accounted for 34.5% of identified MRSA cases in this study.

As a Conformité Européene-approved in vitro diagnosticproduct, the LightCycler MRSA Advanced Test has been validatedwith an inclusive collection of MRSA strains. A previous evalua-tion study performed in the United States reported that the Light-Cycler MRSA Advanced Test had a sensitivity of 95.2% (13).However, the epidemiologically relevant clones frequently en-countered in certain geographic regions may not be congruentwith the spectrum of variant isolates circulating in other regions.In this study, we found that the characteristic SNPs that inducedthe Tm shift in the LightCycler MRSA Advanced Test were presentin more than one-third of MRSA clones prevalent in the HongKong region. When the PCR results were interpreted solely basedon the target range of melting temperature (Tm) values (57.0 to62.0°C) defined by the manufacturer, the LightCycler MRSA Ad-vanced Test exhibited diagnostic sensitivity of 48.9%. However,regardless of the postamplification Tm values, the overall sensitiv-ity of the test could be improved to 83.3% without compromisingthe specificity. From a technical point of view, although positiveamplifications with atypical Tm values of 55°C can be manuallyinterpreted individually as positive, the statement of “peak(s) out-

FIG 1 Multiple sequence alignment of the selected SCCmec-orfX junction of various MRSA clones. type-I, type I SCCmec (GenBank accession no. AB033763);type-II, type II SCCmec (GenBank accession no. D86934); type-III, type III SCCmec (GenBank accession no. AB047089); type-IV, type IV SCCmec (GenBankaccession no. AB425823); type-IV1, type IVa SCCmec (GenBank accession no. AB063172); type-IV2, type IVb SCCmec (GenBank accession no. AB063173);type-IV3, type IVc SCCmec (GenBank accession no. AB096217); type-IVg, type IVg SCCmec (GenBank accession no. DQ106887); type-V, type V SCCmec(GenBank accession no. AB121219); type-VI, type VI SCCmec (GenBank accession no. AF411935); type-VII, type VII SCCmec (GenBank accession no.AB373032); 921015-ST45-55C, 921032-ST45-55C, 128050-ST45-55C, and 921103-ST45-55C, CC45 lineage MRSA clones with Tm values of 55°C; 127700-ST398-55C, CC398 lineage MRSA clones with Tm values of 55°C; 128359-ST672-55C, CC672 lineage MRSA clones with Tm values of 55°C; 128505-ST8-59C,CC8 lineage MRSA clones with Tm values of 59°C; 128390-ST22-59C, CC22 lineage MRSA clones with Tm values of 59°C. Numbers indicate nucleotide positions,asterisks indicate nucleotide positions showing consensus among all the selected SCCmec-orfX sequences, and the boxes indicate the nucleotide substitutions.

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side target TM range” reported by the automated assay interpre-tation software might confuse users who are not familiar with thetest. The interpretation procedures also may be complicated inregions with high prevalence rates of different lineages of MRSAclones. We recommend that the manufacturer amend the inter-pretation algorithm in order to extend the detection coverage ofthe kit for different lineages of MRSA clones.

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